Pharmaceutical composition comprising antisense-nucleic acid for prevention and/or treatment of neuronal injury, degeneration and cell death and for the treatment of neoplasms

ABSTRACT

A pharmaceutical composition comprising an effective amount of a compound which is capable from preventing and treating neuronal injury, degeneration, cell death and/or neoplasms in which expression of c-jun, c-fos or jun-B plays a causal role which compound being an antisense nucleic acid or effective derivative thereof, said antisense nucleic acid hybridizing with an area of the messenger RNA (mRNA) and/or DNA encoding c-jun, c-fos or jun-B.

This is a 371 of PCT/EP94/02218, filed Jul. 10, 1993.

The present invention is related to a pharmaceutical composition and adiagnostic agent comprising an effective amount of a compound which iscapable of preventing and treating neuronal injury, cell death and/orneoplasms in which expression of c-jun, c-fos or jun-B plays a causalrole, particularly, antisense nucleic acid or -oligonucleotideshybridizing with an area of the messenger RNA (mRNA) and/or DNAcomprising the genes for c-jun, c-fos or jun-B; the use of the compoundfor the preparation of a pharmaceutical composition for the treatment ofneoplasms and/or the prevention and/or treatment of neuronal injury anddegeneration related with the expression of c-jun, c-fos or jun-B.

Schlingensiepen et al. report in Proceedings of the American Associationfor Cancer Research, Vol. 32, p. 303, Abstract No. 1799, 82. AnnualMeeting of the American Association for Cancer Research, Houston, USA,1991 that c-jun and jun-B genes share high sequence homology with thev-jun gene. They belong to the immediate early gene group. C-juntogether with c-fos constitutes the DNA binding factor AP-1. C-jun andjun-B expression was inhibited in different cell lines usingphosphorothioate oligodeoxynucleotides. C-jun inhibition stronglyreduced 3H-thymidine incorporation in two mammary carcinoma cell lines,in the rat phaeochromocytoma cell line PC-12 and in NH 3T3 mousefibroblasts. The inhibition of c-jun expression and of c-fos expressionhad very similar effects in the same cell lines inhibition of jun-Bexpression drastically increases 3H-thymidine uptake to more than 10fold. 10-jun is meant to have the characteristics of a proto-oncogenebut jun-B appears to be an anti-oncogene with strong anti-proliferativeaction similar to that of p53. The results suggest that jun-B and c-junto be functional antagonists with regard to their effect on cell growth.This investigation was carried out in order to elucidate the function ofrespective genes and proteins. This abstract does not suggest anytherapeutic concept.

From the Journal of Cellular Biochemistry, Abstract B 977, KeystoneSymposia on Molecular & Cellular Biology, 1993, Schlingensiepen et al.report of two homologues of the proto-oncogene c-jun which have beenidentified in mammals. In that abstract it is speculated that jun-B mayplay a role in cell-differentiation. In order to investigate functionalquestions of the jun-B gene antisense phosophorothioateoligo-deoxynucleotides (S-ODN) have been used to specifically inhibitexpression of c-jun and jun-B in neuronally differentiating PC-12 tumorcells in primary neuronal cell cultures from the rat hippocampus.Western blot analysis revealed specific reductions in the respective Junprotein levels by more than 90% after application of 2 μM S-ODN. Inneuronal cell cultures neurite outgrowth was strongly inhibited afterinhibition of jun-B expression but was enhanced after application ofanti c-jun-S-ODN. Even more drastic changes were observed in neuronallydifferentiating PC-12 tumor cells. The results suggest that jun-B playsa crucial role in cell differentiation while c-jun appears to inhibitdifferentiation. A therapeutic concept is also not available from thatdisclosure.

From Biomedicine & Pharmacotherapy, Abstract 38, from the 5.International Congress on Differentiation Therapy, Schlingensiepen etal. report also about the results published in Journal of CellularBiochemistry.

In Developmental Genetics 14: 305-312 (1993) Schlingensiepen et al.report about the induction of the jun-B and/or c-jun transcriptionfactors. The induction is part of the immediate early response todiverse stimuli that induce alterations in cellular programs. In orderto determine functional significance of the jun-B and/or c-juntranscription antisense phosphorothioate oligodeoxynucleotides were usedto inhibit the expression of the genes in proliferating and neuronallydifferentiating cells. In cell culture studies it was found thatinhibition of jun-B expression markedly reduced morphologicaldifferentiation. Conversely, inhibition of c-jun proteins synthesisenhanced morphological differentiation of both primary neurons and PC-12tumor cells.

EP-A-0 305 929 deals with membranes with bound oligonucleotides andpeptides directly bound onto the membrane. The method for synthesizingoligonucleotides directly bound onto a membrane provides a means forgenerating membrane affinity supports. A modified membrane for themethod of direct synthesis is also provided.

WO 92/15680 deals with a method and compositions for the selectiveinhibition of gene expression. Disclosed are methods and compositionsfor the selective inhibition gene expression through the application ofantisense RNA technology. Antisense RNA constructs employ the use ofantisense intron DNA corresponding to distinct intron regions of thegene whose expression is targeted for down-regulation. In an exemplaryembodiment a human lung cancer cell line (NCI-H460a) with a homozygousspontaneous K-ras mutation was transfected with a recombinant plasmidthat synthesizes a genomic segment of K-ras in antisense orientation.Translation of the mutated K-ras m RNA was specifically inhibited,whereas expression of H-ras and N-ras was unchanged. A three-fold growthinhibition occurred in H460a cells when expression of the mutated rasp21 protein was down-regulated by antisense RNA and cells remainedviable. The growth of H460a tumors in nu/nu mice was substantiallyreduced by expressed K-ras antisense RNA.

Dan Mercola in Prospects for Antisense Nucleic Acid Therapy of Cancerand AIDS, pp. 83-114, 1991 deals with the use of antisense fos RNA and,to a lesser extent, antisense jun RNA. Such antisense RNA hascontributed to understanding of the roles of gene products in cell cyleregulation, differentiation and so on. Progess in the application ofantisense RNA and oligonucleotides to these topics and implications fordiagnostic and therapeutic approaches are considered.

S. van den Berg in Prospects for Antisense Nucleic Acid Therapy andCancer and AIDS, pp. 63-70, 1991 deals with antisense fosoligodeoxyribonucleotides suppressing the generation of chromosomalaberrations. The fast induction of the expression product FOS nuclearonco-protein by serum treatment of starved cells was used to test thefunctional stability of antisense oligodeoxyribonucleotides. Unmodifiedoligodeoxyribonucleotides lost their blocking effect with a half-life ofabout 2 hours, modification of the backbone by thioesters extended thehalf-life to about 4 hours. The modified oligodeoxyribonucleotides whereused to unravel a decisive role of FOS in a complex physiologic event:The induction of chromosomal aberrations upon overexpression ofoncogenes like ras and mos and upon irradiation of fibroblasts withUV-light.

Induction of the c-Fos, Jun-B and/or c-Jun transcription factors is partof the immediate early response to diverse stimuli that inducealterations in cellular programs. C-jun and c-fos are proto-oncogeneswhose expression is required for induction of cell proliferation whilethe function of the Jun-B transcription factor has remained unclear.

Neuronal cell injury and cell death due e. g. to hypoxia or hypoglycemiamay occur in cause of responses of the cell to diverse stimuli inducingalterations in cellular programs.

It is an object of the present invention to provide a pharmaceuticalcomposition for the prevention and/or treatment of neuronal injuryand/or cell death. Surprinsingly, the expression of the c-fos and c-jungene plays a causal role in neuronal cell injury and cell death due e.g. to hypoxia or hypoglycemia.

Furthermore, surprisingly, expression of the Jun-B protein is requiredfor the differentiation of normal and neoplastic cells and inhibition ofc-Jun protein expression enhances the differentiation of such cells.Based on that result the present invention provides a pharmaceuticalcompositon for the treatment of neoplasms by enhancing jun-B expressionand/or inhibiting c-jun expression.

A pharmaceutical composition comprising antisense nucleic acids oreffective derivatives thereof which hybridize with an area of the mRNAsor DNA comprising the genes for c-jun, c-fos or jun-B are able to solvethe problems addressed above. The antisense nucleic acid is able tohybridize with regions of the c-jun, jun-B or c-fos mRNAs. It isunderstood by the skilled person that fragments of the antisense nucleicacids and antisense nucleic acids containing these sequences workaccording to the invention so long as production of the c-Jun and/orc-Fos and/or Jun-B proteins is reduced or inhibited.

According to the invention the antisense-oligonucleotides are obtainableby solid phase synthesis using phosphite triester chemistry by growingthe nucleotide chain in 3′-5′ direction in that the respectivenucleotide is coupled to the first nucleotide which is covalentlyattached to the solid phase comprising the steps of

-   -   cleaving 5′DMT protecting group of the previous nucleotide,    -   adding the respective nucleotide for chain propagation,    -   modifying the phosphite group subsequently cap unreacted        5′-hydroxyl groups and    -   cleaving the oligonucleotide from the solid support,    -   followed by working up the synthesis product.

The chemical structures of oligodeoxy-ribonucleotides are given in FIG.1 as well as the respective structures of antisenseoligo-ribonucleotides are given in FIG. 2. The oligonucleotide chain isto be understood as a detail out of a longer nucleotide chain.

In FIG. 1 lit. B means an organic base such as adenine (A), guanine (G),cytosine (C) and thymine (T) which are coupled via N9 (A,G) or N1 (D,T)to the desoxyribose. The sequence of the bases is the reverse complementof the genetic target sequence (mRNA-sequence). The modifications usedare

-   -   1. Oligodeoxy-ribonucleotides where all R¹ are substituted by    -   1.1 R¹=O    -   1.2 R¹=S    -   1.3 R¹=F    -   1.4 R¹=CH₃    -   1.5 R¹=OEt    -   2. Oligodeoxy-ribonucleotides where R¹ is varied at the        internucleotide phosphates within one oligonucleotide

-   -   where B=deoxy-ribonucleotide dA, dC, dG or dT depending on gene        sequence        -   p=internucleotide phosphate        -   n=an oligodeoxy-ribonucleotide stretch of length 6-20 bases    -   2.1 R^(1a)=S; R^(1b)=O    -   2.2 R^(1a)=CH₃; R^(1b)=O    -   2.3 R^(1a)=S; R^(1b)=CH₃    -   2.4 R^(1a)=CH₃; R^(1b)=S    -   3. Oligodeoxy-ribonucleotides where R¹ is alternated at the        internucleotide phosphates within one oligonucleotide

-   -   -   where B=deoxy-ribonucleotide dA, dC, dG or dT depending on            gene sequence            -   p=internucleotide phosphate            -   n=an oligodeoxy-ribodinucleotide stretch of length 4-12                dinucleotides

    -   3.2 R^(1a)=S; R^(1b)=O

    -   3.2 R^(1a)=CH₃; R^(1b)=O

    -   3.3 R^(1a)=S; R^(1b)=CH₃

    -   4. Any of the compounds 1.1-1.5; 2.1-2.4; 3.1-3.3 coupled at R²        with the following compounds which are covalently coupled to        increase cellular uptake

    -   4.1 cholesterol

    -   4.2 poly(L)lysine

    -   4.3 transferrin

    -   4.4 folic acid

    -   5. Any of the compounds 1.1-1.5; 2.1-2.4; 3.1-3.3 coupled at R³        with the following compounds which are covalently coupled to        increase cellular uptake

    -   5.1 cholesterol

    -   5.2 poly(L)lysine

    -   5.3 transferrin

    -   5.4 folic acid

In the case of the RNA-oligonucleotides (FIG. 2) are the basis (adenine(A), guanine (G), cytosine (C), uracil (U)) coupled via N9 (A,G) or N1(C,U) to the ribose. The sequence of the basis is the reverse complementof the genetic target sequence (mRNA-sequence). The modifications in theoligo-nucleotide sequence used are as follows

-   -   6. Oligo-ribonucleotides where all R¹ are substituted by    -   6.1 R¹=O    -   6.2 R¹=S    -   6.3 R¹=F    -   6.4 R¹=CH₃    -   6.5 R¹=OEt    -   7. Oligo-ribonucleotides where R¹ is varied at the        inter-nucleotide phosphates within one oligonucleotide

-   -   -   where B=ribonucleotide A, C, G or T depending on gene            sequence            -   p=internucleotide phosphate            -   n=an oligo-ribonucleotide stretch of length 4-20 bases

    -   7.1 R^(1a)=S; R^(1b)=O

    -   7.2 R^(1a)=CH₃; R^(1b)=O

    -   7.3 R^(1a)=S; R^(1b)=CH₃

    -   7.4 R^(1a)=CH₃; R^(1b)=S

    -   8. Oligo-ribonucleotides where R¹ is alternated at the        internucleotide phosphates within one oligonucleotide

-   -   -   where B=ribonucleotide A, C, G or T depending on gene            sequence            -   p=internucleotide phosphate            -   n=an oligo-ribodinucleotide stretch of length 4-12                dinucleotides

    -   8.2 R^(1a)=S; R^(1b)=O

    -   8.2 R^(1a)=CH₃; R^(1b)=O

    -   8.3 R^(1a)=S; R^(1b)=CH₃

    -   9. Any of the compounds 6.1-6.5; 7.1-7.4; 8.1-8.3 coupled at R²        with the following compounds which are covalently coupled to        increase cellular uptake

    -   9.1 cholesterol

    -   9.2 poly(L)lysine

    -   9.3 transferrin

    -   10. Any of the compounds 6.1-6.5; 7.1-7.4; 8.1-8.3 coupled at R³        the following compounds are covalently coupled to increased        cellular uptake

    -   10.1 cholesterol

    -   10.2 poly(L)lysine

    -   10.3 transferrin

    -   11. Any of the compounds 6.1-6.5; 7.1-7.4; 8.1-8.3; 9.1-9.3;        10.1-10.3 where all R⁴ are substituted by

    -   11.1 R⁴=O

    -   11.2 R⁴=F

    -   11.3 R⁴=CH₃

In a preferred embodiment the c-jun antisense nucleic acid comprisingthe sequences as identified in the sequence listing, Seq. ID. No. 1-55and 174-177.

In a preferred embodiment the jun-B antisense nucleic acids iscomprising the sequences as identified in the sequence listing Seq. IDNo. 56-97 and 178, 179.

In another preferred embodiment the c-fos antisense nucleic acid iscomprising the sequences as identified in the sequence listing underSeq. ID No. 98-173 and 180-185.

It is possible that one single individual sequence as mentioned aboveworks as an antisense nucleic acid or oligo-nucleotide structureaccording to the invention. However, it is also possible that one strandof nucleotides comprises more than one of the sequences as mentionedabove directly covalently linked or with other nucleotides covalentlylinked inbetween. Preferably, individual oligonucleotides of thesequences as outlined in the sequence listing are addressed.

The sequence5′GTCCCTATAC GAAC 3′(SEQ IN NO: 186)served as randomized control sequence.

In a preferred embodiment of these oligo-nucleotides they arephosphorotioate derivatives.

Modifications of the antisense-oligonucleotides are advantageous sincethey are not as fast destroyed by endogeneous factors when applied asthis is valid for naturally occuring nucleotide sequences. However, itis understood by the skilled person that also naturally occuringnucleotides having the disclosed sequence can be used according to theinvention. In a very preferred embodiment the modification is aphosphorothioate modification.

The synthesis of the oligodeoxy-nucleotide of the invention is describedas an example in a greater detail as follows.

Oligodeoxy-nucleotides were synthesized by stepwise 5′-addition ofprotected nucleosides using phosphite triester chemistry. The nucleotideA was introduced as5′dimethoxy-trityl-deoxyadenosine(N-benzoyl)-N,N′-diisopropyl-2-cyano-ethylphosphoramidite (0.1 M); C was introduced by a5′-dimethoxytrityl-deoxycytidine(N⁴-benzoyl)-N,N′-diisopropyl-2-cyanoethylphosphoramidite; G was introduced as5′-dimethoxy-trityl-deoxyguanosine(N⁸-isobutyryl)-N,N′-diisopropyl-2-cyanoethylphosphoramidite and the T was introduced as5′-dimethodytrityl-deoxythymidine-N,N′-diisopropyl-2-cyanoethylphosphoramidite. The nucleosides were preferably applied in 0.1 Mconcentration dissolved in acetonitrile.

Synthesis was performed on controlled pore glass particles ofapproximately 150 μm diameter (pore diameter 500 Å) to which the most 3′nucleoside is covalently attached via a long-chain alkylanin linker(average loading 30 μmol/g solid support).

The solid support was loaded into a cylindrical synthesis column, cappedon both ends with filters which permit adequate flow of reagents buthold back the solid synthesis support. Reagents were delivered andwithdrawn from the synthesis column using positive pressure of inertgas. The nucleotides were added to the growing oligonucleotide chain in3′→5′ direction. Each nucleotide was coupled using one round of thefollowing synthesis cycle:

Cleave 5′DMT (dimethoxytrityl) protecting group of the previousnucleotide with 3-chloroacetic acid in dichloro-methane followed bywashing the column with anhydrous acetonitrile. Then simultaneously oneof the bases in form of their protected derivative depending on thesequence was added plus tetrazole in acetonitrile. After reaction thereaction mixture has been withdrawn and the phosphite was oxidized witha mixture of sulfur (S₈) in carbon disulfid/pyridine/-triethylamine.After the oxidation reaction the mixture was withdrawn and the columnwas washed with acetonitrile. The unreacted 5′-hydroxyl groups werecapped with simultaneous addition of 1-methylimidazole and aceticanhydryide/lutidine-/tetrahydrofuran. Thereafter, the synthesis columnwas washed with acetonitrile and the next cycle was started.

The work up procedure and purification of the synthesis products occuredas follows.

After the addition of the last nucleotide the deoxynucleotides werecleaved from the solid support by incubation in ammonia solution.Exocyclic base protecting groups were removed by further incubation Inammonia. Then the ammonia was evaporated under vacuum. Full-lengthsynthesis products still bearing the 5′DMT protecting group wereseparated from shorter failure contaminants using reverse phase highperformance liquid chromatography on silica C₁₈ stationary phase.Eluents from the product peak were collected, dried under vacuum and the5′-DMT protecting group cleaved by incubation in acetic acid which wasevaporated thereafter under vacuum. The synthesis products weresolubilized in the deionized water and extracted three times withdiethylether. Then the products were dried in vacuo. Another HPLC-AXchromatography was performed and the eluents from the product peak weredialysed against excess of Trisbuffer as well as a second dialysisagainst deionized water. The final products were lyophilized and storeddry.

The antisense nucleic acids of the invention are intermediate productsof the pharmaceutical composition or medicament of the invention. Thismedicament can be used for treating and/or preventing neuronal celldeath, for treating neoplasms in which the expression of c-jun and/orjun-B or c-fos is of relevance for the pathogenicity. The pharmaceuticalcomposition may comprise besides the effective compound(s) suitablecarrier agents, solvents and other ingredients known in the art forproducing medicaments. Preferably, these agents facilitate theadminstration of the pharmaceutical composition of the invention.Typically, the pharmaceutical composition is administered as i.v.infusion or i.v. bolus injection. The amount of the active ingredient tobe adminstered is typical in the range of 0.2-50 mg of theoligonucleotide per kg body weight per day, in particular 1-12 mg/kgbody weight per day.

The effect of antisense oligo-nucleotides specific for c-jun, jun-B andc-fos on protection against neuronal cell death was investigated. It wasdemonstrated that that c-fos as well as c-jun play a causal role inneuronal cell death. Also the role of these gene in the differentiationand proliferation of neoplastic cells was investigated. It wasdemonstrated that inhibition of c-Jun protein synthesis could enhancedifferentiation of neoplastic cells. It was demonstrated that antisenseoligodeoxynucleotides as well as phosphorothioate modified nucleicacids, complementary to the mRNAs of c-jun, jun-B and c-fos specificallyinhibit expression of the respective proteins.

In principal the compound which can be used as an active compound in thephamaceutical composition can be used as a diagnostic tool forevaluating whether the respective genes are exprersses. Typically, aradio active label nucleotides are hybridized by the method of northernblotting with is well-known in the art or in situ with a sample to beexamined. The degree of hybridization is a measure for the degree ofexpression of the respective genes.

FIG. 3

Western blot analysis of rat PC-12 cell lysates. Effects of differentphosphorothioate oligodeoxynucleotides on c-Fos protein expression.Incubation time with oligodeoxynucleotide were 6 h. Lane 1: randomizedcontrol S-ODN; Lane 2: anti-c-fos S-ODN-180; Lane 3: anti-c-fosS-ODN-182. 10 μg of total protein were used per lane.

FIG. 4

Effects of different phosphorothioate oligodeoxynucleotides on c-Jun andJun-B protein expression. A: Western blots of NIH 3T3 cell lysate probedwith an anti-c-jun antibody. B:SK-BR3 cell lysates, probed with ananti-jun-B antibody.

Incubation times with oligodeoxynucleotide were: Lanes 1-3: 6 h; Lanes4-6: 24 h. Lanes 1 and 4: randomized control S-ODN; Lanes 2 and 5:anti-jun-B S-ODN-62; Lane 3 and 6: anti-c-jun S-ODN-13. 10 μg of totalprotein were used per lane.

FIG. 5

Effects of different phosphorothioate oligodeoxynucleotides on c-Jun,Jun-B and c-Fos protein expression.

-   A: Enzyme-linked immunosorbent assay of rat PC-12 cell lysates    incubated with c-jun (rat specific) antisense oligo-deoxynucleotides    174, 175, 176, 177.-   B: Enzyme-linked immunosorbent assay of human SK-Br-3 cell lysates    incubated with c-jun (human-specific) antisense    oligodeoxynucleotides 1, 7, 13, 17, 20, 23, 26, 31, 31, 39, 45, 51    or 54.-   C: Enzyme-linked immunosorbent assay of rat PC-12 cell lysates    incubated with jun-B (rat-specific) antisense oligo-deoxynucleotides    178 or 179.-   D: Enzyme-linked immunosorbent assay of human SK-Br-3 cell lysates    incubated with jun-B (human-specific) antisense    oligodeoxynucleotides 57, 62, 64, 69, 80 85, 89, 92, 95 or 97.-   E: Enzyme-linked immunosorbent assay of rat PC-12 cell lysates    incubated with c-fos (rat-specific) antisense oligo-deoxynucleotides    180, 181, 182, 183, 184 or 185.-   F: Enzyme-linked immunosorbent assay of human SK-Br-3 cell lysates    incubated with c-fos (human-specific) antisense oligonucleotides 98,    99, 102, 103, 108, 116, 121, 130, 139, 144, 152, 158, 165, 170 or    173.

Phosphorothioate-oligodeoxynucleotides were used at 2 μM concentration.Control cells were left untreated (white bars) or treated with 2 μM ofrandomized control phosphorothioate oligonucleotides (grey bars).

FIG. 6

Survival of rat cerebellar neurons following hypoxia. Phosphorothioateoligonucleotides were used at 1 μM concentration. Control cells were notsubjected to hypoxia (white bar). Hypoxia control cells were either nottreated with oligonucleotide (black bar, C) or treated with the sameconcentration of randomized control phosphorothioateoligo-deoxynucleotide (grey bar). Error bars correspond to 1 SD.

FIG. 7

Enhanced proliferation arrest after suppression of c-Jun proteinsynthesis and lack of proliferation arrest in NGF treated PC-12 cellsafter suppression of Jun-B protein synthesis. PC-12 cell number after 8days of NGF treatment. Bars represent the mean of 4 values. Grey bars: 2μM randomized control S-ODN; White bars: 2 μM anti-c-jun S-ODN-174;Black bars: 2 μM anti-jun-B S-ODN-179. Error bars correspond to 1 SD.

FIG. 8

Morphological differentiation of NGF treated PC-12 cells afterinhibition of c-jun or jun-B protein synthesis.

-   A: Control cells not treated with phosphorothioate    oligo-deoxynucleotides.-   B: Cells incubated with 2 μM anti-jun-B S-ODN-179.-   C: Cells incubated with 2 μM anti-c-jun S-ODN-174.

The invention is further explained by he following non-limitingexamples.

EXAMPLE 1 Cell Lines and Proliferation Assays

NIH 3T3 mouse fibroblasts and SK-Br-3 human mammary carcinoma cells weregrown in RPMI medium (Gibco) supplemented with 100 U/ml penicillin, 100μg/ml streptomycin, 5% FCS. PC-12 rat phaeochromocytoma cells were grownin Dulbecco's modified Eagle's medium (DMEM medium Seromed),supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 5% FCS.

EXAMPLE 2 Western Blot

Cells were kept under low serum conditions in RPMI/2% FCS for 3 days,trypsinized and preincubated in RPMI/5% FCS/2 μM S-ODN for 5 min. 3×10⁶cells were plated into 260 ml culture flasks and grown for the timesindicated in RPMI/5% FCS/2 μM S-ODN, trypsinized, spun down and lysed byfreezing. SDS-polyacrylamide gel electrophoresis, blotting andchemiluminescence detection were performed according to standardtechniques. Blots were probed with a rabbit anti mouse-c-jun antibody(Oncogene Science) or with a rabbit anti human-jun-B antibody (OncogeneScience) or with a rabbit anti-c-fos antibody (Oncogene Science), usinggoat anti-rabbit IgG-alkaline-phosphatase conjugate (BoehringerMannheim) as second antibody and CSPD (Tropix) for chemiluminescentdetection.

EXAMPLE 3 Enzyme-Linked Immunosorbent Assay (ELISA)

Cell lysates were diluted in 50 mM carbonate buffer at pH 9.0 andimmobilized on immunon II plates (Dynatech Laboratories, Inc.)overnight. Antigen solution was removed and 200 μl/well phosphatebuffered saline (PBS)/1% BSA/0.02% azide were added to blocknon-specific protein binding. Following incubation at room temperaturefor 2 h solution was removed. After washing with PBS plates were airdried for 3 h. Specific antibodies for c-jun, jun-B or c-fos (Oncogene,Santa Cruz, Biotechnology Inc.) were added at 50 μl/well, diluted inblocking buffer. Following 1 h incubation at room temperature sampleswere removed and subsequently wells were washed four times withPBS/0.05% Tween 20. Then 50 μl of secondary antibody-phosphataseconjugate were added and removed after 1 h. Wells were washed withdiethanolamine buffer (10 mM diethanolamine, 0.5 mM MgCl₂, pH 9.5). 1tablet of Sigma 104 phosphatase substrate was dissolved in 5 mldiethanolamine buffer. 50 μl of the substrate solution were added perwell. The reaction was stopped with 50 μl 0.1 M EDTA (pH 7.5) and plateswere read on a microtitration plate reader.

EXAMPLE 4 Neuronal Survival

Cerebella were removed from the brains of 8 day old rats under sterileconditions and were transferred into 0.1% trypsin, 0.1% DNase inphosphate buffered saline/glucose solution for 15 min at 20° C.,followed by 1.5% soybean trypsin inhibitor (Sigma) for 5 min. Cells weredissociated in a mixture of Dulbecco's modified Eagle's medium and Ham'sF-12 medium (50%/50%, v/v; DMEM F-12, Gibco) supplemented with KCl 25mM, penicillin (5 U/ml), gentamycin (5 μg/ml) and 30 mM glucose. Cellswere centrifuged at 300 x g for 3 min, and resuspended in the samemedium, supplemented with 10% fetal calf serum (Gibco). Cells wereplated in 3 cm dishes (0.5 ml per well) coated with poly-L-lysine (10μg/ml, Sigma) to a density of 1×10⁵ cells/well and transferred to anincubator with humidified atmosphere with 95% O₂/5% CO₂. Cytosinearabinoside (40 μM) was added after 24 h to inhibit glial cellproliferation. On day 16 after seeding, cells were exposed to anoxia for16 h by placing them in a hermetic chamber containing a humidifiedatmosphere with 95% N₂/5% CO₂. The chamber was transferred into anincubator at 37° C. Phosphorothioate oligodeoxynucleotides were added at1 μM concentration 8 h before the onset of anoxia. Neuronal cell injurywas determined 26 h later by staining with trypan blue dye exclusion(incubation with 0.4% trypan blue for 5 min).

EXAMPLE 5 Proliferation of PC-12 Cells After Treatment with NGF andDifferent Phosphorothioate Oligodeoxynucleotides.

PC-12 cells were plated at a density of 2,500 cells/well in DMEM(Seromed) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin,5% FCS/2 μM S-ODN. 2 μM S-ODNs were added 6 h after plating. 24 h afterplating, cells were incubated with 10 ng/ml of the 2.5 S subfraction ofnerve growth factor (NGF) (Boehringer Mannheim) for 8 days. Cell numberwas determined by using trypan blue dye exclusion (incubation with 0.4%trypan blue for 5 min) and counting of cells in a Neubauer countingchamber.

EXAMPLE 6 PC-12 Tumor Cell Differentiation

PC-12 cells were plated at a density of 2,500 cells/well (Seromed) into96 well microtitration plates coated with poly-L-lysine (10 μg/ml,Sigma) in 100 μl of DMEM supplemented with 100 U/ml penicillin, 100μg/ml streptomycin, 5% FCS, S-ODNs were added at 2 μM concentration 2 hafter plating. 6 h after plating, cells were incubated with 40 ng/ml ofthe 2.5 S subfraction of nerve growth factor (NGF) (Boehringer Mannheim)for 11 days.

1. An antisense oligonucleotide, wherein the antisense oligonucleotide is SEQ ID NO: 2, the antisense oligonucleotide being, optionally, substituent-modified, or phosphorothioated, or substituent-modified and phosphorothioated.
 2. A composition for human administration comprising the antisense oligonucleotide of claim 1 together with a physiologically acceptable carrier. 